15 Semduramicin sodium

15 Semduramicin sodium

15 Semduramicin sodium O O NaO OH HO O H O H OH O O O O H O O H O OH H H Semduramicin sodium C45H75O16Na MW: 895.1 CAS No.: 119068-77-8 Semduramicin C45H76O16 MW: 873.1 CAS No.: 113378-31-7 [Summary of semduramicin sodium] Semduramicin is a polyether antibiotic obtained by the incubation of Actinomadura roseorufa, and has the chemical structure shown above. The one used as a feed additive is its sodium salt (SD). For physicochemical properties, SD technical occurs as white to gray-white crystalline powder. It is soluble in methanol, sparingly soluble in ethanol, slightly soluble in dichloromethane and in ether, and practically insoluble in water and in isooctane.

Formulations containing not less than 0.5 % of semduramicin are designated as deleterious substances under the Cabinet Order for the Designation of the Poisonous and Deleterious Substances (Cabnet Order No.2, 1965). For the handling of these substances, make sure to comform to the procedures specified in the Poisonous and Deleterious Substances Act (Act No.303, 1950). SD has an antibacterial effect on part of the Gram-positive bacteria and a growth promoting effect on chickens (including broilers). «Standards and specifications in the Act on Safety Assurance and Quality Improvement of Feeds» SD is a pure-grade antibiotic that was designated as a feed additive as of July 18, 1994.

The specifications for feeds containing this ingredient are specified in Appended Table 1, 1-(1)-C of the Ministerial Ordinance Concerning the Ingredient Specifications for Feeds and Feed Additives. (in g(potency)/t) Added amount 25 25 Feed of interest Starting chicks Growing chicks Starting broilers Growing broilers For chickens (except for broilers) For broilers Like SL, NR, MN and LS, excessive consumption of SD can cause growth disturbace in chickens. It is therefore necessary to strictly conform to the specified amounts of addition (25 g (potency) per 1 ton

of the feed of interest) and to achieve homogeneous mixture to secure the safety. For this reason, feed manufacturers are required to control the chicken feeds that contain SD according to the separately described control test method (6 Chiku B No.1012, notified by the Head of the Livestock Industry Bureau, the Ministry of Agriculture, Forestry and Fisheries, as of July 18, 1994). [Methods listed in the Feed Analysis Standards] 1 Quantitative test method - Plate method 1.1 Premix [Feed Analysis Standards, Chapter 9, Section 2, 15.1.1] Scope of application: Premix not containing TP A. Reagent preparation 1) Dilution solvent: A mixture of water and methanol (7:3) 2) Semduramicin standard solution.

Dry a suitable amount of semduramicin working standard[1] under reduced pressure (not exceeding 0.67 kPa) at 100°C for 3 hours, weigh accurately not less than 40 mg, accurately add methanol and dissolve to prepare a semduramicin standard stock solution with a concentration of 1 mg (potency)/mL[2] .

At the time of use, accurately dilute a quantity of the standard stock solution with the dilution solvent to prepare high- and low-concentration standard solutions with concentrations of 2.5 and 1.25 µg (potency)/mL, respectively[3] . 3) Culture medium: Medium F-15 4) Bacterial suspension and amount of addition. Use Bacillus subtilis ATCC 6633 as the test organism. Add about 0.1 mL of a spore suspension with a concentration of 1×109 spores/mL per 100 mL of the culture medium 100 mL. 5) Agar plate. Proceed by the agar well method. 6) Extracting solvent: A mixture of dichloromethane[4] and 2,2,4-trimethylpentane (1:1) B.

Preparation of sample solution Extraction. Weigh accurately 3 to 5 g of the analysis sample, place in a 200-mL stoppered Erlenmeyer flask, add 100 mL of the extracting solvent, extract with stirring for 20 minutes, and filter the extract through filter paper (No.5A). Dilute a quantity of the filtrate 2- to 10-fold with the extracting solvent[5] , and use as the sample solution subject to column treatment. Column treatment. Wash a silica gel minicolumn (690 mg) with 5 mL of methanol and with 5 mL of dichloromethane 5 mL in this order.

Accurately transfer a quantity of the sample solution (equivalent to 0.1 to 0.5 mg (potency) as SD) to the minicolumn and allow to flow down until the amount in the minicolumn reservoir reaches 1 mL. Add accurately 20 mL of a mixture of dichloromethane[4] and acetone (9:1) and accurately 8 mL of a mixture of dichloromethane[4] and acetone (4:1)[6] to the minicolumn, and allow to flow out in this order to wash the minicolumn. Place a 100-mL round-bottom flask under the minicolumn, and add 10 mL of a mixture of acetone and methanol (4:1) to the minicolumn to elute SD. Evaporate the eluate into dryness in a water bath at 50°C, and accurately add a quantity of the dilution solvent to dissolve the residue.

Accurately dilute a

quantity of of this liquid with the dilution solvent to prepare high- and low-concentration sample solutions with concentrations of 2.5 and 1.25 µg (potency)/mL, respectively. C. Quantification[7] Proceed by the 2-2 dose method[8] . «Summary of analysis method» This method is intended to determine the amount of SD in a premix by microbiological assay (2-2 dose method) using a sample solution prepared by extracting with a mixture of dichloromethane and 2,2,4-trimethylpentane (1:1) and purifying through a silica gel minicolumn. The ratio of the concentration of the high-concentration standard solution to that of the low-concentration standard solution is set to 2 because the slope of the standard response line is relatively steep.

Except for TP, none of the antibacterial substances approved for combined use with SD interfere with the quantification of SD.

The flow sheet of this method is shown in Figure 9.2.15-1. Sample (3.0-5.0 g) Extract with 100 mL of dichloromethane-2,2,4-trimethylpentane (1:1). (magnetic sitter, 20 min) Filter (filter paper: No.5A). Wash the minicolumn with 20 mL of dichloromethane-acetone (9:1). Wash the minicolumn with 8 mL of dichloromethane-acetone (4:1). Evaporate into dryness under reduced pressure (in a water bath at 5°C). Dissolve the residue with 20-25 mL of water-methanol (7:3). Dispense to agar plates (allow to stand at 10-20°C). Incubate (35-37°C for 16-24 hr).

Measure the inhibition zone diameter. Calculate the potency by the 2-2 dose method.

Load a quantity of the filtrate onto a silica gel minicolumn (previously washed with 5 mL of methanol and 5 mL of dichloromethane). Dilute a quantity of the solution with water-methanol (7:3) to prepare high- and low-concentration sample solutions (2.5 and 1.25 µg(potency)/mL, i l ) Elute SD with 10 mL of acetone-methanol (4:1) (into a 200-mL round-bottom Figure 9.2.15-1 Quantitative test methods for semduramicin sodium (premix) References: Shoichi Yamatani: Research Report of Animal Feed, 21, 113 (1996) History in the Feed Analysis Standards [18] New

«Validation of analysis method» Spike recovery and repeatability Chicken premix 1 0.4~10 3 100.7~103.7 7.0 Chicken premix 2 0.4~10 3 98.0~100.3 1.8 Chicken premix 3 0.4~10 3 98.3~101.0 1.7 Sample type Repeat Spike concentration (g(potency)/kg) Spike recovery (%) Repeatability RSD(% or less) Collaborative study Chicken premix 8 2 99.2 2.1 3.6 No. of labs Sample type Spike concentration (g(potency)/kg ) Spike recovery (%) Intra-lab repeatability RSDr(%) Inter-lab reproducibility RSDR(%) «Notes and precautions» [1] For the difinition etc. of semduramicin working standard, refer to «Notes and precautions» [9] in Section 1, 1 of this Chapter.

[2] For the method of preparation for the standard stock solution, refer to «Notes and precautions» [10] in Section 1, 1 of this Chapter. Method of preparation: example (when the weighed amount is 50 mg) If the labeled potency of the working standard is 951 µg (potency)/mg, 50 mg of the working standard contains 47,550 µg (potency) (i.e., 50 mg × 951 µg (potency)/mg. To prepare a standard stock solution with a concentration of 1,000 µg (potency)/m, the required amount of solvent is thus calculated to be 47.55 mL (i.e., 47,550 µg (potency) / 1,000 µg (potency)/mL. Therefore, completely transfer 50 mg of the working standard to an Erlenmeyer flask containing 47.55 mL of methanol and dissolve to prepare a standard stock solution with a concentration of 1,000 µg (potency)/mL.

[3] For method of preparation for the standard solution, refer to «Notes and precautions» [8] in Section 1, 1 of this Chapter.

An example method of preparation semduramicin standard solution is shown in Table 9.2.15-1. Table 9.2.15-1 Method of preparation for semduramicin standard solution (premix, example) Test tube No. 1 2 3 4 Amount (mL) of standard solution 2 2 5 5 18 18 15 5 Concentration (µg(potency)/mL) 100 10 2.5 1.25 Note: "2 mL" means "2 mL of standard stock solution (1 mg(potency)/mL)". Amount (mL) of water-methanol (7:3) [4] The dichloromethane shall be of JIS Guaranteed grade. Although there are some dichloromethane formulations spiked with about 0.3% methanol as a stabilizing agent for testing residual pesticides and PCB, the use of such grades affect the retention of the column and results in a poor recovery.

[5] Exa ples of the dilution factor for the filtrate, the amount loaded onto the column , etc. are shown in Table 9.2.15-2.

[6] The amount of the mixture of dichloromethane and acetone (4:1) shall be precisely 8 mL. The larger the amount, the lower the recovery. Table 9.2.15-2 Dilution factor for the filtrate, amount loaded onto the column, etc. 2 2 10 20 5 10 4 10 25 10 50 10 10 25 20 Note: the concentration of the residue in the solution prepared by diluting the residue with dilution solvent in a flask. Dilute this solution further with dilution solvent to prepare high and low-concentration sample solutions with concentrations of 2.5 and 1.25 µg(potency)/mL, respectively.

Amount of sample collected (equivalent to mg(potency)) Dilution factor for filtrate (-fold) Amount loaded onto column (mL) Amount of dissolved residue (mL) Concentration of solution (µg(potency)/mL) [7] An example standard response line for SD is shown inFigure 9.2.15-2.

The ratio of the concentration of the high-concentration standard solution to that of the low- concentration standard solution is set to 2 because the slope of the standard resonse line is relatively steep. Figure 9.2.15-2 Standard response line for semduramicin (premix, example) (Bacillis subtilis ATCC 6633, Medium F-15, Agar well method) [8] Refer to «Notes and precautions» [53] to [60] in Section 1, 1 of this Chapter. 1.2 Feed [Feed Analysis Standards, Chapter 9, Section 2, 15.2.1] Scope of application : Feed not containing TP 25 20 15 Corrected inhibition zone diameter (mm) Concentration of semduramicin (µg (potency)/mL) 0.5 1 2 4 10

A. Reagent preparation 1) Dilution solvent: A mixture of water and methanol (7:3) 2) Semduramicin standard solution. Dry a suitable amount of semduramicin working standard under reduced pressure (not exceeding 0.67 kPa) at 100°C for 3 hours, weigh accurately not less than 40 mg, accurately add methanol and dissolve to prepare a semduramicin standard stock solution with a concentration of 1 mg (potency)/mL. At the time of use, accurately dilute a quantity of the standard stock solution with dilution solvent to prepare high- and low-concentration standard solutions with concentrations of 2.5 and 1.25 µg (potency)/mL, respectively[1] .

3) Culture medium: Medium F-15 4) Bacterial suspension and amount of addition. Use Bacillus subtilis ATCC 6633 as the test organism. Add about 0.1 mL of the spore suspension with a concentration of 1×109 spores/mL per 100 mL of the culture medium. 5) Agar plate. Proceed by the agar well method. B. Preparation of sample solution Extraction. Weigh accurately a quantity of the analysis sample (equivalent to 0.5 mg (potency) as SD)[2] , place in a 200-mL stoppered Erlenmeyer flask, add 100 mL of a mixture of dichloromethane[3] and 2,2,4- trimethylpentane (1:1), and extract with stirring for 20 minutes.

Transfer the extract to a 50-mL stoppered centrifuge tube, centrifuge at 650×g for 15 minutes, and use as the sample solution subject to column treatment.

Column treatment. Wash a silica gel minicolumn (690 mg) with 5 mL of methanol and 5 mL of dichloromethane in this order. Transfer accurately 25 mL of the sample solution to the minicolumn, and allow to flow down until the amount remaining in the reservoir reaches 1 mL. Accuraetly add 20 mL of a mixture of dichloromethane[3] and acetone (9:1) and 8 mL of a mixture of dichloromethane and acetone (4+1)[4] to the minicolumn, and allow to flow out in this order to wash the minicolumn. Place a 100-mL round-bottom flask under the minicolumn, add 10 mL of a mixture of acetone and methanol (4:1) to the minicolumn to elute SD.

Evaporate the eluate into dryness in a water bath at 50°C, and accurately add 25 mL of the dilution solvent to dissolve the residue. Accurately dilute a quantity of this liquid with the dilution solvent to prepare high- and low-concentration sample solutions with concentrations of 2.5 and 1.25 µg (potency)/mL, respectively.

C. Quantification[5] Proceed by the 2-2 dose method[6] . «Summary of analysis method» This method is intended to determine the amount of SD in a feed by microbiological assay using a sample solution prepared by extracting with a mixture of dichloromethane and 2,2,4-trimethylpentane (1:1) and purifying through a silica gel minicolumn. The ratio of the concentration of the high- concentration standard solution to that of the low-concentration standard solution is set to 2 as the slope

of the standard response line is relatively steep. This method is almost the same as the quantitative test method for SD in a premix.

Except for TP, none of the antibacterial substances approved for combined use with SD interfere with the quantification of SD. The flow sheet of this method is shown in Figure 9.2.15-3. Sample (usually 20 g, equivalent to 0.5 mg(potency) as SD) Extract with 100 mL of dichloromethane-2,2,4-trimethylpentane (1:1). (magnetic stirrer, 20 min) Centrifuge (at 650×g for 15 min). Wash the silica gel minocolumn with 20 mL of dichloromethane-aceton (9:1). Wash the silica gel minicolumn with 8 mL of dichloromethane-acetone (4:1). Elute SD with 10 mL of acetone-methanol (4:1) (into a 200-mL round-bottom fla Evaporate into dryness under reduced pressure (in a water bath at 50°C).

Dissolve the residue in 25 mL of water-methanol (7:3). Dispense to agar plates (allow to stand at 10-20°C for 2 hr). Incubate (at 35-37°C for 16-24 hr).

Measure the inhibition zone diameter. Calculate the potency by the 2-2 dose method. Load 25 mL of the supernatant liquid onto a silica gel minicolumn (previously washed with 5 mL of methanol and 5 mL of dichloromehtane). Dilute a quantity of the solution with water-methanol (7:3) to prepare high- and low-concentration sample solutions (2.5 and 1.25 µg(potency)/mL, i l ) Figure 9.2.15-3 Quantitative test methods for semduramicin sodium (feed not containing TP) References: Shoichi Yamatani: Research Report of Animal Feed, 19, 155 (1994) History in the Feed Analysis Standards [18] New «Validation of analysis method» Spike recovery and repeatability Starting chick formula feed 25~50 3 99.3~107.0 11.0 Starting broiler formula feed 25~50 3 97.7~105.0 3.4 Growing broiler formula feed 25~50 3 98.3~99.7 8.5 Sample type Repeat Spike concentration (g(potency)/t) Spike recovery (%) Repeatability RSD(% or less)

Collaborative study Growing broiler formula feed 7 25 100.3 3.8 5.2 No. of labs Sample type Spike concentration (g(potency)/t) Spike recovery (%) Intra-lab repeatability RSDr(%) Inter-lab reproducibility RSDR(%) «Notes and precautions» [1] For the method of preparation for the standard solution, refer to «Notes and precautions» [8] in Section 1, 1 of this Chapter. An example method of preparation for the semduramicin standard solution is shown in Table 9.2.15-3. Table 9.2.15-3 Method of preparation for semduramicin standard solution (feed that does not contain TP, example) Test tube No. 1 2 3 4 Amount (mL) of standard solution 2 2 5 5 18 18 15 5 Concentration (µg(potency)/mL) 100 10 2.5 1.25 Note: "2 mL" means "2 mL of standard stock solution (1 mg(potency)/mL)".

Amount (mL) of water-methanol (7:3) [2] Usually corresponds to 20 g. [3] The dichloromethane shall be of JIS Guaranteed grade. Although there are some dichloromethane formulations spiked with about 0.3% methanol as a stabilizing agent for testing residual pesticides and PCB, the use of such grades affect the retention of the column and results in a poor recovery. [4] The amount of the mixture of dichloromethane and acetone (4:1) shall be accurately 8 mL. The larger the amount, the lower the recovery.

[5] An example standard response line for SD is shown in Figure 9.2.15-4. [6] Refer to «Notes and precautions» [53] to [60] in Section 1, 1 of this Chapter. Figure 9.2.15-4 Standard response line for semduramicin (feed that does not contain TP, example) (Bacillis subtilis ATCC 6633, Medium F-15, Agar well method) 25 20 15 0.5 1 2 4 10 Corrected inhibition zone diameter (mm) Concentration of semduramicin (µg (potency)/mL)

2 Quantitative test method for polyether antibiotics by liquid chromatography 2.1 Premix [Feed Analysis Standards, Chapter 9, Section 2, 15.1.2] Antibiotics of interest: SL, SD, NR and MN (4 components) A.

Reagent preparation 1) Salinomycin sodium standard solution. Dry a suitable amount of salinomycin working standard[1] under reduced pressure (not exceeding 0.67 kPa) at 60°C for 3 hours, weigh accurately a quantity equivalent to 20 mg (potency), place in a 100-mL volumetric flask, add methanol to dissolve, and further add methanol up to the marked line to prepare a salinomycin sodium standard stock solution (1 mL of this solution contains an amount equivalent to 0.2 mg (potency) as salinomycin sodium). At the time of use, accurately dilute a quantity of standard stock solution with a mixture of methanol and water (9:1) to prepare several salinomycin sodium standard solutions containing salinomycin sodium in amounts equivalent to 2.5 to 20 µg (potency) in 1 mL.

2) Semduramicin sodium standard solution. Weigh accurately a quantity of semduramicin working standard equivalent to 20 mg (potency)[1] , place in a 100-mL volumetric flask, add methanol to dissolve, and further add methanol up to the marked line to prepare a standard stock solution (1 mL of this solution contains an amount equivalent to 0.2 mg (potency) as semduramicin sodium). At the time of use, accurately dilute a quantity of the standard stock solution with methanol to prepare several semduramicin sodium standard solutions containing semduramicin sodium in amounts equivalent to 2.5 to 20 µg (potency) in 1 mL.

3) Narasin standard solution. Weigh accurately a quantity of narasin working standard equivalent to 20 mg (potency)[1] , place in a 100-mL volumetric flask, add methanol to dissolve, and further add methanol up to the marked line to prepare a narasin standard stock solution (1 mL of this solution contains narasin in an amount equivalent to 0.2 mg (potency)). At the time of use, accurately dilute a quantity of the standard stock solution with a mixture of methanol and water (9:1) to prepare several narasin standard solutions containing narasin in amounts equivalent to 0.5 to 20 µg (potency) in 1 mL.

4) Monensin sodium standard solution. Weigh accurately a quantity of monensin working standard equivalent to 20 mg (potency)[1] , place in a 100-mL volumetric flask, add methanol to dissolve, and further add methanol up to the marked line to prepare a monensin sodium standard stock solution (1 mL of this solution contains an amount equivalent to 0.2 mg (potency) as monensin sodium). At the time of use, accurately dilute a quantity of the standard stock solution with a mixture of methanol and water (9:1) to prepare several monensin sodium standard solutions containing monensin sodium in amounts equivalent to 2.5 to 20 µg (potency) in 1 mL.

B. Quantification Extraction. Weigh accurately 2 to 5 g of the analysis sample, place in a 200-mL stoppered Erlenmeyer flask, add 100 mL of a mixture of methanol and water (9:1), extract with stirring for 20 minutes, and filter the extract through filter paper (No.5A). Accurately dilute a quantity of the filtrate with a mixture of methanol and water (9:1), filter through membrane filter (pore size not exceeding 0.5 µm), and use the filtrate as the sample solution subject to liquid chromatography.

Liquid chromatography. Inject 20 µL each of the sample solution and antibiotic standard solutions into a liquid chromatograph to obtain chromatograms. Example operating conditions Detector: Ultraviolet-visible absorption detector (measured wavelength: 520 nm) Column: Octadecylsilanized silica gel column (4.6 mm in internal diameter, 150 mm in length, 5 µm in particle size)Note 1 [2] Eluent: A mixture of methanol, water and acetic acid (940:60:1) Reaction solutionNote 2 : Gradually add 10 mL of sulfuric acid to 475 mL of methanol while stirring, add 15 g of vanillin and dissolve (prepare at the time of use).

Flow rate: 0.6 mL/min for the eluent; 0.6 mL/min for the reaction solution Reaction vessel temperature: 95°C Calculation. Calculate the peak height or peak area from the obtained chromatogram[3] to prepare the calibration curve, and estimate the amount of each antibioticNote 3, 4 .

Note 1. Use a Mightysil RP-18 GP (Kanto Chemical Co., Inc.) or an equivalent. 2. Develop by allowing the eluate from the column to react with the reaction solution through the reaction coil (0.5 mm in internal diameter, 5 mm in length (10 m )) in the reaction vessel, and immediately transfer to the ultraviolet-visible absorption detector. The reaction solution shall be used in a light-resistant container. 3. For monensin sodium, the calculated amount of monensin A shall be regarded as the amount of monensin sodium. The peak of monensin A appears as the main peak on the chromatogram from each monensin sodium standard solution.

On the chromatogram of the sample solution, the peak of monensin A appears at the same retention time as the peak of monensin A from the standard solution.

4. For narasin, the calculated amount of narasin A shall be regarded as the amount of narasin. The peak of narasin A appears as the main peak on the chromatogram of each narasin standard solution. On the chromatogram from the sample solution, the peak of narasin A appears at the same retention time as the peak of narasin A from the standard solution. «Summary of analysis method» This method is intended to determine the amount of salinomycin, semduramicin, narasin A, and monensin A in a premix by determining the absorbances of their derivatives produced by extracting with a mixture of methanol and water (9:1), separating by liquid chromatography using an octadecylsilanized silica gel (ODS) column, and allowing to react with vanillin.

It is also called the post-column derivatization method.

The principle of this derivatization (chromogenic) reaction depends on the so-called Komarowsky reaction, which involves aldol condensation of the alcoholic hydroxyl groups of salinomycin, semduramicin, narasin A and monensin A with the benzaldehyde group of vanillin, in an acidic solution containing sulfuric acid, to produce derivatives of these antibiotics that have wavelengths of maximal absorption of about 520 nm.

This method allows for simultaneous quantification of salinomycin sodium (SL), semduramicin sodium (SD), narasin (NR), and monensin sodium (MN).

Care should be taken that, of the peaks of monensin sodium, the peak of monensin B can appear at the same retention time as the peak of semduramicin sodium, and thus interfere with the quantification. For reference, the nature of separation of the mixed standard solution is shown in Figure 9.3.1-1. Because of the above-mentioned possibility of interference from peaks other than those of interest, it is preferable to use a single-component standard solution rather than a mixed standard solution for the preparation of the calibration curve.

Figure 9.3.1-1 Chromatogram for a mixed standard solution (SL: 100 ng, SD: 200 ng, NR-A: 100 ng, MN-A: 50 ng) Narasin is a mixture of narasin A, narasin B, narasin D and narasin I, and the “narasin” designated as a feed additive is defined as containing narasin A as the main ingredient. Monensin is a mixture of monensin A, monensin B, monensin C and monensin D, and the “monensin” designated as a feed additive is defined as containing monensin A as the main ingredient. In the test method described here, the quantified amounts of narasin A and of monensin A are regarded as the amounts of narasin and of monensin, respectively, based on the premises that commercial narasin and monensin formulations contain narasin and monensin at a concentration of not less than 95%, respectively.

It should be borne in mind, therefore, that the “narasin” and “monensin” quantified by this method are not exactly the same as the “narasin” and “monensin” quantified by microbiological assay.

For more details, refer to «Notes and precautions» [1] of General Notice 13 in Chapter 1. The flow sheet of this method is shown in Figure 9.3.1-2. SD MN SL NR 10 SD derivative NR-A derivative SL derivative MN-A derivative 10 Retention time (min) Absorbance

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